Thioredoxin (Trx) is a redox-active protein that plays a fundamental role in the cellular responses to oxidants, heavy metals, and alkylating agents. The Trx system scavenges peroxides, repairs damaged proteins, provides bases for the DNA repair machinery, and facilitates the expression of protective genes through the reduction of key transcription factors. Trx is mainly localized to the cytoplasm, but translocates to the nucleus in response to toxic and inflammatory stimuli. However, the factors regulating this redistribution are unknown. In addition to the two active site cysteines (Cys), Trx contains three other Cys residues, the function of which is unknown. This proposal seeks to test the hypothesis that oxidation of Trx signals its redistribution to the nucleus. This will be possible due to the recent development of the Redox Western blot technique, which measures the redox state of the active site and structural Cys residues in Trx. The first aim of this proposal is to test the hypothesis that oxidants in general induce the translocation of Trx to the nucleus, and that this is associated with an oxidation of Trx. The second aim is to determine whether oxidation of Trx is an event that is common to known inducers of Trx translocation (cytokines, phorbol esters, ultraviolet light and ionizing radiation, cobalt chloride, and hypoxia). The third aim is to identify the amino acid residues that are necessary for translocation of Trx to the nucleus. To accomplish this, each of the five Cys residues will be individually mutated to Serines via site-directed mutagenesis, and the subcellular localization of the mutant Trx will be assessed before and after exposure to oxidants or other inducers of nuclear translocation. Successful completion of these aims will define the redox state of nuclear and cytoplasmic Trx in cells exposed to toxic and inflammatory stimuli, and will show whether thiol oxidation contributes to the dynamic control of Trx Iocalization within cells. This information will provide the basis for future investigations into the role of nuclear Trx in redox-dependent processes such as transcriptional up-regulation of protective genes.